The present invention relates to the length of telomeres and to their effect on proliferation and senescence in cells. More specifically, it concerns the ability of hnRNP A1 and its shortened derivative UP1 to alter the length of telomeres in cells. More precisely, the invention relates to the ability of A1/UP1 to bind telomerase RNA, to bind and to protect mammalian telomeric DNA, and to modulate telomere extension and replication. Finally, the present invention relates to agents which can interfere with the binding of A1/UP1 to telomeres and telomerase, and to the use of protection, extension and replication assays to measure the biological impact of these agents.
Telomeres are the DNA structure at the ends of the chromosomes of eukaryotes, including human, and are comprised of variable lengths of double-stranded repeats terminating with single-stranded G-rich repeats originally identified in yeast and protozoa (McElligot and Wellinger, 1997, EMBO J. 16:3705).
Review articles concerning telomeres include Greider, 1996, (Ann. Rev. Biochem. 65:337). Relevant articles on various aspects of telomeres include Muller et al., 1991, Cell 67:815; Yu et al., 1991, Cell 67:823; Gray et al., 1991, Cell 67:807; de Lange, 1995, xe2x80x9cTelomere Dynamics and Genome Instability in Human Cancerxe2x80x9d, E. Blackburn and C. W. Greider (eds), in Telomeres, Cold Spring Harbor Laboratory Press, pp. 265-293; Rhyu, 1995, J. Nati. Cancer Inst. 87:884; Greider and Harley, 1996, xe2x80x9cTelomeres and Telomerase in Cell Senescence and Immortalizationxe2x80x9d, in Cellular Aging and Cell Death, Wiley-Liss, Inc., pp. 123-138. Thus, telomeres are involved in the maintenance of chromosome structure and function. Furthermore, it appears that loss of telomeric DNA activates cellular processes involved in the detection and control of DNA damage, and affects cellular proliferation and senescence.
Maintenance of the integrity of telomeres is essential for cell survival (Sandell et al., 1993, Cell 75:729-739). The proliferative potential of cells has been correlated with alterations in the length of these tandemly repeated sequences (Counter et al., 1992, EMBO J. 11:1921-1929). In addition, maintenance of telomere length and the regulation thereof are essential, pluripotent cellular functions as they are involved in the transmission of genetic information to daughter cells, senescence, cell growth and cancer (Blackburn, 1992, Annu. Rev. Biochem. 61:113-129).
The finite replicative capacity of normal human cells, e.g., fibroblasts, is characterized by a cessation of proliferation in spite of the presence of serum growth factors. This cessation of replication after a maximum of 50 to 100 population doublings in vitro is referred to as cellular senescence. See, Goldstein, 1990, Science 249:1129. The replicative life span of cells is inversely proportional to the in vivo age of the donor (Martin et al., 1979, Lab. Invest. 23:86) and is therefore suggested to reflect in vivo ageing on a cellular level.
Cellular immortalization (unlimited life span) may be thought of as an abnormal escape from cellular senescence. Normal human somatic cells appear to be mortal, i.e., have finite replication potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultures in vitro appear to require the aid of transforming oncoproteins to become immortal and even then the frequency of immortalization is 10xe2x88x926 to 10xe2x88x927 (Shay et al., 1989, Exp. Cell Res. 184:109). A variety of hypotheses have been advanced over the years to explain the causes of cellular senescence. One such hypothesis proposes that the loss of telomeric DNA with age, eventually triggers cell cycle exit and cellular senescence (Harley et al. 1990, Nature (London) 345:458-460; Allsopp et al., 1992, Proc. Natl. Acad. Sci. USA 89:10114-10118; Counter et al., 1992, EMBO J. 11:1921-1929).
Human primary fibroblasts in culture enter crisis after a precise number of cell division associated with gradual telomere shortening, at which point all the cells die (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885). Mouse primary fibroblasts have longer and/or more stable telomeres and display a similar behavior when cultured in vitro. However, after crisis, primary mouse cells in culture spontaneously immortalize with a frequency of 10xe2x88x926, possibly because longer telomeres facilitate the growth of mutant cells (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885).
It should be noted, as mentioned above, that other hypotheses have been advanced to explain senescence and that there is yet to be a consensus or a universally accepted hypothesis therefor. Previously, the causal relationship between telomeres and cancer/ageing/senescence had been built entirely on correlative studies.
Recent data has shown that telomeres play a direct role in cell senescence and transformation. Indeed, Wright et al., 1996, EMBO J. 15:1734-1741, using telomerase-negative cells which have limited life span in tissue culture, have shown that the introduction of oligonucleotides carrying telomeric repeats causes telomere elongation and increases the proliferative capacity of these cells. Moreover, the authors state that xe2x80x9cprevious studies had shown a remarkable correlation between telomere length and cellular senescence. The present results provide the first experimental evidence for a true causal relationship between telomere length and a limited proliferative capacityxe2x80x9d. Feng et al., 1995 (Science 269:1236-1241) showed that a human cell line (HeLa) transfected with an antisense telomerase RNA, looses telomeric DNA and begins to die after 23-26 cell doublings. The authors claim that xe2x80x9cthe results support the hypothesis that telomere loss leads to crisis and cell death once telomeres are shortened to a critical lengthxe2x80x9d.
The telomerase is part of a multi-component ribonucleoprotein complex. The RNA component of the human telomerase ribonucleoprotein has been identified. The catalytic protein subunit has recently been cloned (Nakamura et al., 1997, Science 277:955).
More recent advances have confirmed the role of telomeres in cell senescence. Overexpression of the catalytic protein component of telomerase can lead to telomere elongation and extension of the proliferative capacity of telomerase-negative fibroblasts in culture (Bodnar et al. 1998, Science 279:349). Overexpression of this protein also prevents the accelerated ageing of human fibroblasts derived from patients with Werner syndrome (Wyllie et al. 2000, Nat. Genet.). Mice and murine ES cells that do not express telomerase RNA show telomere shortening and become impaired in long-term viability (Lee et al. 1998, Nature 392:569; Niida et al. 1998, Nat. Genet. 19:203). Recent studies have also supported the role of telomeres in cellular transformation. The expression of a catalytically inactive form of telomerase or the inactivation of telomerase RNA in human immortal and cancer cell lines promotes telomere shortening, growth arrest and cell death (Hahn et al. 1999, Nat. Med. 5:1164; Herbert et al. 1999, Proc. Natl. Acad. Sci. USA 96:14276; Zhang et al. 1999, Genes Dev. 13:2388).
The length of telomeres and cell viability can also be affected by proteins that bind to vertebrate telomeres. TRF1 and TRF2 are proteins that bind to double-stranded telomeric repeats. Overexpression of TRF1 promotes telomere shortening (van Steensel and de Lange 1997, Nature 385:740). Expression of a dominant negative version of TRF2 promotes end-to-end fusion of chromosomes, an event which leads to p53-dependent cell death by apoptosis (van Steensel et al. 1998, Cell 92:401; Karlseder et al. 1999, Science 283:1321).
The postulated link between senescence/proliferation of cells and telomere length has led to therapeutic and diagnostic methods relating to telomere length or to telomerase, the ribonucleoprotein enzyme involved in the synthesis of telomeric DNA. PCT Publication No. 93/23572 describes oligonucleotide agents that either reduce the loss of telomeric sequence during passage of cells in vitro, or increase telomeric length of immortal cells in vitro. The same type of approach is also taught in PCT Publication No. 94/13383 and U.S. Pat. No. 5,484,508 which refer to methods and compositions for the determination of telomere length and telomerase activity, as well as to methods to inhibit telomerase activity in the treatment of proliferative diseases. Methods to increase or decrease the length of telomeres through an action on telomerase is also taught. The agents which are shown to reduce telomere loss of telomere length during proliferation are oligonucleotides which promote synthesis of DNA at the telomere ends, as well as telomerase.
PCT Publication No. 95/13383 discloses a method and compositions for increasing telomeric length in normal cells so as to increase the proliferative capacity of the cells and to delay the onset of cellular senescence. PCT Publication No.96/10035 teaches that telomere length serves as a biomarker for cell turnover. Furthermore, it discloses that measurement of telomere length can be used to diagnose and stage cancer and other diseases as well as cell senescence.
PCT publication WO98/11204 teaches two nucleic acid sequences termed TPC2 and TPC3 and amino acid sequences of the polypeptides encoded thereby which can be used to detect regulators of telomere length and telomerase activity in mammalian cells. TPC3 is shown to regulate telomerase activity and telomere length.
PCT publication WO98/11207 teaches telomerase reporter constructs to be used in assessing the transcriptional activity of mammalian telomerase gene transcription regulatory region. This application also relates to the use of these constructs to identify agents which modulate transcription of the telomerase gene.
Proteins that bind mammalian telomeric repeats, either to double-stranded repeats or single-stranded repeats, are also targets for telomere length regulation.
U.S. Pat. No. 5,733,730 and PCT WO97/08314 relate to the double-stranded DNA binding factor TRF and discloses a method of purifying telomerase from mammalian cells.
PCT Publication No. WO 98/00537 relates to the single-stranded DNA binding factor A1/UP1 and discloses methods and compositions to increase or decrease telomeric length. A1 is a member of the abundant family of heterogeneous nuclear ribonucleoprotein particles (hnRNP) proteins (Dreyfuss et al. 1993. Ann. Rev. Biochem. 62:289). There are over 20 such hnRNP proteins in human cells. HnRNP A1 can modulate telomere length once introduced into a mouse cell line (WO98/00537; and La Branche et al., 1998. Nat. Genet. 19:199-202). Thus, UP1 lacks the last N-terminal 124aa of A1 (the glycine-rich domain), but shares with A1 the first 196aa. The first 196aa comprises two RNA Recognition Motifs (RRMs); RRM1 extending from aa 15-93, and RRM2 extending from aa 106-184. UP1 can modulate telomere length once introduced into a mouse or a human cell line (WO98/00537; and La Branche et al., 1998., supra).
Telomeres are essential for normal cellular function, by preventing degradation and aberrant recombination of chromosome termini and facilitating the complete replication of chromosome ends. Vertebrate telomeres contain variable numbers of TAGGGT-repeats in double-stranded form and terminate with a single-stranded overhang of the G-rich strand, the strand making the 3xe2x80x2 end of the chromosome. The ribonucleoprotein enzyme telomerase directs the synthesis of telomeric repeat units onto this G-rich strand, thereby counteracting the loss of sequence that occurs at each cell division. It is thought that the G-rich strand will then serve as substrate for the synthesis of the complementary strand by DNA primase followed by conventional DNA polymerases (Greider and Blackburn, 1989, Nature 337:331; Greider, 1996 supra).
The presence of a 3xe2x80x2 overhang of the G-rich strand suggests that single-stranded DNA binding activities will play an important role in telomere function. Proteins that can bind to single-stranded telomeric repeats include protein xcex1 of Oxytricha, Stylonychia and Euplotes. The 56 kD protein xcex1 of Oxytricha exists as an heterodimer with the 41 kD protein xcex2. These proteins protect single-strand overhangs from nuclease digestion and chemical modification (Fang and Cech 1995, in Telomeres, Blackburn, E. H., and Greider, C. W., eds, pp. 69-105, Cold Spring Harbor Press, Cold Spring Harbor, N. Y.; Gray et al., 1991, supra). Moreover, the binding of an xcex1/xcex1 homodimer or an xcex1/xcex2 heterodimer to telomeric DNA renders the end inaccessible to telomerase (Froelich-Ammon et al., 1998, Genes Dev 12:1504). The Chlamydomonas protein Gbp1p binds to single-stranded G-rich telomeric DNA (Johnston et al., 1999, Mol Cell Biol 19: 923), but its role in telomere function in vivo remains to be shown. While telomerase RNA makes direct contacts with single-stranded extensions during repeat synthesis, proteins components of Tetrahymena and Euplotes telomerases can also interact with telomeric single-stranded DNA substrates by protein-DNA interactions (Gandhi and Collins, 1998, Genes Dev 12:721).
In Saccharomyces cerevisiae, Est1p and Est4p/Cdc13p have properties of terminus-binding proteins and their association with G-rich extensions may mediate recognition by telomerase. Mutant strains engineered not to express Est1p, or expressing mutated forms of Cdc13p, undergo telomere attrition despite having wild-type levels of telomerase (Nugent et al., 1996, Science 274:249; Virta-Pearlman, et al.,1996, Genes Dev 10:3094). While Est1p interacts with telomerase RNA in vitro and in vivo, its presence is not essential in some telomerase activity assays in vitro (Steiner et al., 1996, Proc Natl Acad Sci U S A 93:2817).
In vertebrates, several proteins can interact with single-stranded G-rich extensions in vitro. However, there has been no demonstration that these proteins bind to telomeres in vivo or that their expression influences the structure of telomeres. Mammalian hnRNP proteins have been reported to associate with RNA and DNA oligonucleotides carrying telomeric repeats. The only mammalian hnRNP protein for which genetic evidence of a function in telomere biogenesis has been obtained is the hnRNP A1 protein. Ectopic expression of A1 promotes telomere elongation in mammalian cells (laBranche et al, supra). Although hnRNP A1 is a well-known modulator of alternative pre-mRNA splicing, several observations are consistent with the notion that the function of A1 is independent of its role in alternative splicing. First, a shortened derivative of A1 (UP1) that has no intrinsic activity in alternative splicing, but which can antagonize the modulatory activity of A1 in splicing extracts, also promotes telomere elongation. Second, UP1 and A1 can interact specifically with single-stranded telomeric repeats in vitro . Third, UP1 may interact with telomerase, as judged by its ability to recover telomerase activity from a cell lysate. While A1 appears to exert its effect on telomeres independently of its function in alternative splicing, it remains to be determined whether A1 has a direct role in telomere biogenesis. It also remains to be determined how the binding of A1/UP1 to telomere modulates telomere biogenesis.
There thus remains a need to modulate the length of telomeres. There also remains a need to identify agents that will enable a modulation of telomere length and/or telomere replication.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
It is thus an aim of the present invention to provide agents and methods to modulate the length and/or replication of telomeres.
The present invention thus relates to a method to recover telomerase from a cell extract comprising a use of the interaction of UP1 with telomerase. More specifically, the invention relates to the demonstration that both A1 and UP1 interact directly and with specificity with the 5xe2x80x2 end of human telomerase RNA (hTR).
Further, the invention relates to the binding of A1/UP1 to telomeric DNA or telomerase RNA. In addition, the invention relates to these specific interactions as valid targets to affect and modulate telomere biogenesis. In one embodiment, preventing the binding of A1 to telomeric DNA in transformed cells, or expressing A1 derivatives that may be affected in binding, can be used to compromise the integrity of telomeres and lead to cell death. In contrast, in another embodiment, improving the binding A1 would be expected to increase telomere length and forestall telomere erosion in some ageing cells.
The invention further relates to the binding of A1/UP1 to telomeric sequences which protects these sequences from nuclease attack (endonuclease and exonucleases). Thus, the present invention provides methods of interfering with A1/UP1 binding to modulate the protective capacity associated with A1/UP1 binding and affect telomere erosion. In another embodiment, the invention relates to a method for preventing A1/UP1 binding in order to produce telomeric ends that are recognized as double-stranded breaks, an event that would lead to rapid cell growth arrest.
The ability of UP1 to recover telomerase activity, and the ability of A1/UP1 to interact with human telomerase RNA suggest that another way by which A1/UP1 may control the length of telomeres is by recruiting telomerase to the ends of chromosomes. Thus, interfering with the binding of A1/UP1 to telomerase RNA would interfere with telomere maintenance and should promote telomere shortening, similar to the effects seen when the activity of telomerase is targeted.
In addition, the present invention relates to binding assays enabling a screening and an identification of agents that modulate A1/UP1 binding to telomerase RNA or telomeric DNA. Also, the present invention aims at providing bindings assays for screening for A1/UP1 derivatives that are affected in their binding abilities.
In one particular embodiment, the invention relates to a method of confirming the biological effect of agents on the binding of A1/UP1 to telomeric DNA or telomerase RNA, or of identifying agents which modulate telomere biogenesis, while not being identified as such by the binding assays of the present invention (e.g. for lack of sensitivity), comprising a testing of the effect of the agent(s) using assays that detect a biological activity which reflects the binding of A1/UP1 to a telomeric DNA or telomerase RNA. In a particular embodiment, the assay is dependent on the binding of A1/UP1 to a DNA sequence carrying multiple telomeric repeats. Non-limiting examples of such assays which detect a biological activity include: (i) an in vitro protection assay in which the binding of A1/UP1 protects against nuclease attack; (ii) an in vitro telomerase extension assay in which extension is inhibited when A1/UP1 binds to the telomeric oligonucleotide; (iii) a terminal nucleotydyl transferase (TdT) elongation assay in which 3xe2x80x2 extension is inhibited by the binding of A1/UP1 to the substrate oligonucleotide; and (iv) a rNTP-dependent DNA polymerase assay in which the binding of A1/UP1 to the telomeric DNA substrate prevents lagging-strand synthesis. It is envisioned that such activity assays might also be used directly to screen for agents without prior testing their effects on DNA binding. Indeed, some agents may not necessarily affect A1/UP1 binding to telomeric DNA (because the assay might not be sensitive enough) but may nevertheless interfere with the activity of A1/UP1, by restoring access of the enzymes (nucleases, telomerase, TdT or polymerase) to the telomeric DNA substrate. These biological assays can also be used to test the activity of derivatives of A1/UP1, as exemplified hereinbelow for the UP1xcex941 and UP1xcex942 derivatives.
Agents, compounds or A1/UP1 derivatives identified in accordance with the present invention as affecting A1/UP1 binding and/or stimulating nuclease, telomerase, TdT or polymerase activity can then be introduced into or incubated with mammalian cells (including human transformed and/or cancer cells), and the effect on telomere structure, and/or cell growth and/or cell viability measured as described above.
While the above procedures are designed to screen for agents, compounds or A1/UP1 derivatives that reduce binding to telomeric DNA or telomerase RNA, such agents, compounds or A1/UP1 derivatives may be identified as enhancers of A1/UP1 binding. In this case, the biological assays described above will confirm their ability to improve protection against nucleases or better prevent access to telomerase, and/or TdT, and/or polymerase. Such agents can be expressed into or incubated with a variety of cells such as senescent, and/or ageing cells in culture, to test whether they improve or maintain telomere integrity during cell growth. Thus, it should be clear that the assays, methods and agents of the present invention relate broadly to modulators (which increase or decrease) of A1/UP1 binding to one of at least its target sequence.
Thus, the present invention also relates to the agents which modulate telomere biogenesis, identified by the methods and assays of the present invention.
In another embodiment of the present invention, the screening assay further comprises an administration of an agent selected as a modulator of telomere biogenesis in vitro, to an animal, tissue, cell type or tumor thereof and a determination of this administration on the animal, tissue, cell type or tumor.
In accordance with the present invention, there is therefore provided a method of identifying an agent which modulates telomere biogenesis in vitro comprising incubating a nucleic acid target sequence for A1/UP1, wherein the target nucleic acid sequence is selected from telomerase RNA and telomeric DNA, together with A1/UP1, a fragment thereof, or a derivative thereof, wherein the A1/UP1, a fragment thereof or derivative thereof is capable of binding to the target sequence; and determining at least one of a binding between said A1/UP1, or fragment, or derivative thereof and the target nucleic acid sequence and an enzymatic activity dependent on the binding between said A1/UP1, or fragment, or derivative thereof and the target nucleic acid sequence; wherein the agent is identified as a modulator of telomere biogenesis when the binding of A1/UP1, or fragment, or derivative thereof or the enzymatic activity is significantly different in the presence of the agent, as compared to in the absence thereof.
There is also provided a method of increasing lagging-strand synthesis on a telomeric sequence in vitro comprising providing of an agent which sequesters A1/UP1, a fragment thereof, or a derivative thereof.
Furthermore, there is provided a method of increasing the activity of DNA polymerase xcex1/primase on a telomeric sequence in vitro, comprising a providing of an agent which sequesters A1/UP1.
There is further provided a method of preventing telomerase extension and/or telomere replication by DNA polymerase xcex1/primase in vitro, comprising increasing the level of A1/UP1, or fragment thereof, or a derivative thereof available for binding to telomere.
In accordance with the present invention, there is also provided a method of maintaining the integrity of telomeric 3xe2x80x2 overhangs comprising a providing of a A1/UP1 telomere binding domain which is capable of binding to the telomeric 3xe2x80x2 overhangs, thereby protecting same from nuclease and/or polymerase activities.
Also in accordance with the present invention, there is also provided a method of protecting single stranded telomeric sequences against nuclease activities, comprising a providing of an A1/UP1 telomere binding domain capable of binding the single stranded telomeric sequences, thereby protecting same from the nuclease activities.
There is also provided a method of identifying an agent which modulates telomere biogenesis comprising a determination of a formation of a ternary complex made up of a telomeric sequence, A1/UP1, or fragment thereof, or fragment thereof and telomerase RNA, wherein a modulator of telomere biogenesis is identified when a level of the formation of the ternary complex is detectably different in the presence of the agent as compared to in the absence thereof.
Also provided is a method of preparing recombinant hnRNPA1 which enables a detectable binding thereto to telomerase RNA.
In accordance with the present invention, there is further provided a method of modulating telomere biogenesis comprising a disturbing of A1/UP1 telomerase RNA interaction.
In accordance with the present invention, there is also provided agents and compositions for modulating telomere biogenesis in vitro identified using an assay or method of the present invention.
In accordance with the present invention, there is also provided a method to identify an agent which modulates telomere biogenesis comprising: providing a telomerase capable of binding with A1/UP1, A1/UP1, derivative or fragment thereof and telomeric DNA, thereby creating a mixture; incubating this mixture with a candidate agent; and assessing a formation of a ternary complex comprising A1/UP1, derivative or fragment thereof, telomerase and telomeric DNA, wherein a modulator of telomere biogenesis is identified when a formulation of the ternary complex is reduced in the presence of the agent as compared to in the absence thereof.
For the purpose of the present invention, the following abbreviations and terms are defined below.
The terminology xe2x80x9cA1/UP1xe2x80x9d relates to hnRNPA1, its derivative UP1 having an activity in telomere biogenesis when in their native form. Non-limiting examples of this activity include binding to its target sequence (e.g. telomere DNA, telomerase RNA), protecting the telomere from nuclease digestion, and affecting lagging-strand synthesis. It will be clear to the skilled artisan (and as exemplified hereinbelow) that recombinants, derivatives or portions of A1/UP1 can also be used and tested in accordance with the present invention.
The terminology xe2x80x9cnucleic acid target sequences for A1/UP1xe2x80x9d, or the like refer to telomere DNA and/or telomerase RNA to which A1/UP1 binds. As exemplified herein, these target sequences can be natural sequences, genetically engineered or synthetically produced. As exemplified herein, a recombinant protein comprising the N-terminal portion of hnRNPA1 up to and including RRM1 can bind specifically to telomeric DNA. Also, a recombinant protein comprising the C-terminal portion of hnRNPA1 up to and including RRM2 is sufficient for specific binding to telomerase RNA.
By xe2x80x9cincreased rate of proliferationxe2x80x9d of a cell, it is meant that a cell has a higher rate of cell divisions compared to normal cells of that cell type, or compared to normal cells within other individuals of that cell type. Examples of such cells but not limited to these, include the CD4+cells of HIV-infected individuals, connective tissue fibroblasts associated with degenerative joint diseases, age-related muscular degeneration, astrocytes associated with Alzheimer""s Disease and endothelial cells associated with atherosclerosis. In each case, one particular type of cell or a group of cells is found to be replicating at an increased level compared to surrounding cells in those tissues, or compared to normal individuals, e.g., individuals not infected with the HIV virus. Thus, the invention features administering to those cells an agent which reduces the loss of telomere length in those cells while they proliferate. The agent itself need not slow the proliferation process, but rather allow the proliferation process to continue for more cell divisions than would be observed in the absence of the agent. The agent may also be useful to slow telomere repeat loss occurring during normal aging, and for reducing telomere repeat loss while expanding cell number ex vivo for cell-based therapies. The agent could thus simply stabilize telomere length.
The assessment of the effect of agents on telomere length modulation or on telomere replication can be assessed by analyzing their effect in modulating the length of telomeres. For example, a particular cell having a known telomere length is chosen and allowed to proliferate and the length of telomere is measured during proliferation. Analysis of telomere length in cells expressing different derivatives or fragments can be identified using methods described below or other methods known to a person of ordinary skill. Non-limiting examples of such derivatives and fragments comprise hnRNP A1 in vitro mutagenized in the RRM1, RRM2 or the glycine-rich domain (see below).
Herein, hnRNP A1 and UP1 are meant to designate the nucleic acid and/or the protein. It will be recognized by a person of ordinary skill whether the protein or nucleic acid fragment is intended.
In related aspects, the present invention features a pharmaceutical composition which include therapeutically effective amounts of modulators of telomere length or replication in accordance with the present invention and pharmaceutically acceptable buffers. In one particular embodiment, these pharmaceutical compositions may include one or more of these inhibitors or agents and can be co-administered with other drugs. For example, AZT is commonly used for treatment of HIV, and may be co-administered with a telomere length reducing agent of the present invention.
In another related aspect, the invention features a method for extending the ability of a cell to replicate. In this method, a replication extending amount of an agent which is active to reduce loss of telomere length within the cell is provided during cell replication. As will be evident to those of ordinary skill in the art, this agent is similar to that useful for treatment of a condition associated with an increased rate of proliferation of a cell. However, this method is useful for the treatment of individuals not suffering from any particular condition, but in which one or more cell types are limiting in that patient, and whose life can be extended by extending the ability of those cells to continue replication. That is, the agent is added to delay the onset of cell senescence characterized by the inability of that cell to replicate further in an individual. One example of such a group of cells includes lymphocytes present in patients suffering from Downs Syndrome (although treatment of such cells may also be useful in individuals not identified as suffering from any particular condition or disease, but simply recognize that one or more cells, or collections of cells are becoming limiting in the life span of that individual).
It is notable that administration of such inhibitors or agents is not expected to be detrimental to any particular individual or animal. However, should gene therapy be used to introduce an agent of the invention into any particular cell population, care should be taken to ensure that the activity of that agent is appropriately regulated, for example, by use of a promoter which can be regulated by the nutrition of the patient. Thus, for example, the promoter may only be activated when the patient eats a particular nutrient, and is otherwise inactive. In this way, should the cell population become malignant, that individual may readily inactivate replication of the cell and cause it to become senescent simply by no longer eating that nutrient.
Another aspect of the present invention features a method for treatment of a condition associated with an elevated level of telomerase activity and/or with longer and/or more stable telomeres within a cell. The method involves administering to that cell a therapeutically effective amount of an agent that reduces or destabilizes the length of the telomeres. The level of telomerase activity can be measured in accordance with the present invention or by any other existing method or equivalent method. Example of such conditions include neoplastic (cancerous) conditions, or conditions associated with the presence of cells which are not normally present in that individual, such as protozoan parasites or opportunistic pathogens. Administration of such an agent can be achieved by any desired mean well known to those of ordinary skill in the art.
By xe2x80x9celevated levelxe2x80x9d of such activity, it is meant that the absolute level of telomerase activity in a particular cell is elevated compared to normal cells in that individual or compared to normal cells in other individuals not suffering from the same condition. The same principle applies to an elevated level or an elevated activity of A1 or UP1 on the length of telomeres.
In addition, the term xe2x80x9ctherapeutically effective amountxe2x80x9d of an inhibitor is a well recognized phrase. The amount actually applied will be dependent upon the individual or animal to which treatment is to be applied, and will preferably be an optimized amount such that an inhibitory effect is achieved without significant side-effects (to the extent that those can be avoided by use of the inhibitor). That is, if effective inhibition can be achieved with no side-effects with the inhibitor at a certain concentration, that concentration should be used as opposed to a higher concentration at which side-effects may become evident. If side-effects are unavoidable, however, the minimum amount of inhibitor that is necessary to achieve the inhibition desired should be used.
By xe2x80x9cinhibitorxe2x80x9d is simply meant any reagent, drug or chemical which is able to inhibit the binding of A1/UP1 to telomeric DNA or telomerase RNA in vivo or in vitro, sufficiently to affect telomere biogenesis. Such inhibitors can be readily identified using standard screening protocols in which A1/UP1 and the nucleic acid is placed in contact with a potential inhibitor and the level of binding is measured in the presence or absence of the inhibitor or in the presence of varying amounts thereof. In this way, not only can useful inhibitors (or stimulators) be identified, but the optimum level of such an inhibitor (or stimulator) can be determined in vitro. Once identified as a modulator in vitro, the agent can be tested in vivo. Numerous methods to test the in vivo effect of this modulator are known to the person skilled in the art to which this application pertains.
As used herein, the terms xe2x80x9cmoleculexe2x80x9d, xe2x80x9ccompoundxe2x80x9d or xe2x80x9cligandxe2x80x9d are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term xe2x80x9cmoleculexe2x80x9d therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling, combinatorial library screening and the like. The terms xe2x80x9crationally selectedxe2x80x9d or xe2x80x9crationally designedxe2x80x9d are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention. As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term xe2x80x9cmoleculexe2x80x9d. For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above. Similarly, in a preferred embodiment, the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain. The molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by a defect in telomere length control or modulation. Alternatively, the molecules identified in accordance with the teachings of the present invention find utility in the development of more efficient modulators of telomerase length.
As used herein, agonists and antagonists of the A1/UP1-target sequence interaction also include potentiators of known compounds with such agonist or antagonist properties. In one embodiment, agonists can be detected by contacting the indicator cell with a compound or mixture thereof or library of molecules (e.g. combinatorial library) for a fixed period time and determining a biological activity as described herein. Of course, antagonists can be similarly detected.
The therapeutic aspect of the invention is related to the now clear observation that the ability of a cell to remain immortal comprises the ability of that cell to maintain or increase the telomere length of chromosomes within that cell. Thus, therapeutic approaches for reducing the potential of a cell to remain immortal focuses on the inhibition of A1 binding, on the level thereof, on a reduction of the protective role of A1/UP1 to nucleases, on a reduction of the recruitment of telomerase by A1/UP1 and the like in those cells in which it is desirable to cause cell death. Example of such cells, but not limited to, include cancerous cells, which are one example of somatic cells which show increased length or stability of telomeres, and have become immortal. The present invention now permits such cells to become mortal once more by a reduction in the size or the stability of the telomeres. As such, inhibition can be achieved in a multitude of ways as, for example, by providing inhibitors, dominant negative mutants, derivatives of these dominant negative mutants and the like.
The inhibitors may be used for treatment of cancers of any type non-limiting examples thereof, including solid tumors and leukemias, carcinoma, histiocytic disorders, leukemia, histiocytosis malignant, Hodgkin""s disease, immunoproliferative small, non-Hodgkin""s lymphoma, plasmacytoma, reticuloendotheliosis, melanoma and the like, osteosarcoma, rhabdomyosarcoma, sarcoma, neoplasms, and for any treatment or of all other conditions in which cells have become immortalized.
In other cases, it is important to slow the loss of telomere sequences, in particular, cells in association with certain diseases (although such treatment is not limited to this, it can be used in normal ageing and ex vivo treatments). For example, some diseases display abnormal fast rate of proliferation of one or more particular groups of cells. One example of such a disease is AIDS, in which death is caused by the early senescence of CD4+cells. It is important to note that such cells age, not because of abnormal loss of telomere sequences (although this may be a factor) but rather because the replicative rate of the CD4+cells is increased such that telomere attrition occurs at a greater rate than normal for that group of cells (Lundblad and Wright, 1996, Cell 87:369). Thus, the present invention provides means to stabilize the length of telomeres. The applicant therefore is providing therapeutic agents which can be used in the treatment of such diseases, and in addition, the means of diagnostic procedures by which similar diseases can be detected so that appropriate therapeutic protocols can be devised and implemented.
Specifically, the loss of telomeres within any particular cell population can be reduced by providing thereto telomere length stabilizing agents, telomere replication stimulators and the like, enhancers of telomerase recruitment to the telomere, xe2x80x9cnuclease inhibitorsxe2x80x9d, according to the present invention. These molecules can be provided within a cell in order to reduce telomere loss or to make that cell immortal. Those of ordinary skill in the art will recognize that other enzymatic activities may be used to enhance the lengthening of telomeres within such cells, for example, by providing certain viral sequences within a cell, non-limiting examples thereof include EBV and SV40. In addition, equivalent such molecules, or other molecules may be readily screened to determine those that will reduce loss of telomeres or stabilize the length of same. Such screening may occur in vitro, and the therapeutic agents discovered by such screening utilized in the above method in vivo. It should be understood that in some situations, in vitro assays such as gel shifts might be sufficient to assess the telomere length stabilizing activity of an agent. In other cases, the assessment of telomere length per se (as opposed to binding of an agent to the telomere) might have to be ascertained in cultured cells for example. The skilled artisan will be able to determine which assay (which are not limited to two listed above) is sufficient to determine the effect of the tested agent on telomere length. As described below, the present invention also provides methods and assays to screen for agents which modulate telomere biogenesis, by assessing an enzymatic activity which is affected by the binding of A1/UP1 to one target sequence thereof (e.g. telomere DNA, telomerase RNA).
Nucleotide sequences are presented herein by single strand, in the 5xe2x80x2 to 3xe2x80x2 direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (1989, Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).
The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
As used herein, xe2x80x9cnucleic acid moleculexe2x80x9d, refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (i.e. genomic DNA, cDNA) and RNA molecules (i.e. mRNA). The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
The term xe2x80x9crecombinant DNAxe2x80x9d as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
The term xe2x80x9cDNA segmentxe2x80x9d, is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
The terminology xe2x80x9camplification pairxe2x80x9d refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods.
Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloningxe2x80x94A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley and Sons Inc., N.Y.).
The term xe2x80x9coligonucleotidexe2x80x9d or xe2x80x9cDNAxe2x80x9d molecule or sequence refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a xe2x80x9cregulatory elementxe2x80x9d according to the present invention, as the term is defined herein. The term xe2x80x9coligonucleotidexe2x80x9d or xe2x80x9cDNAxe2x80x9d can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5xe2x80x2 to 3xe2x80x2 direction. xe2x80x9cNucleic acid hybridizationxe2x80x9d refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989, supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65xc2x0 C. with a labeled probe in a solution containing 50% formamide, high salt (5xc3x97SSC or 5xc3x97SSPE), 5xc3x97Denhardt""s solution, 1% SDS, and 100 xcexcg/ml denatured carrier DNA (i.e. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2xc3x97SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42xc2x0 C. (moderate stringency) or 65xc2x0 C. (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 1989, supra).
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and xcex1-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5xe2x80x2 ends of the probes using gamma 32P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (i.e. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
As used herein, xe2x80x9coligonucleotidesxe2x80x9d or xe2x80x9coligosxe2x80x9d define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods.
As used herein, a xe2x80x9cprimerxe2x80x9d defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Qxcex2 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86,1173-1177; Lizardi et al., 1988, Bio Technology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably, amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S. Patent are incorporated herein by reference). In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss, 1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).
As used herein, the term xe2x80x9cgenexe2x80x9d is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A xe2x80x9cstructural genexe2x80x9d defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
A xe2x80x9cheterologousxe2x80x9d (i.e. a heterologous gene) region of a DNA molecule is a subsegment segment of DNA within a larger segment that is not found in association therewith in nature. The term xe2x80x9cheterologousxe2x80x9d can be similarly used to define two polypeptidic segments not joined together in nature. Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, xcex2-galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.
The term xe2x80x9cvectorxe2x80x9d is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
The term xe2x80x9cexpressionxe2x80x9d defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
The terminology xe2x80x9cexpression vectorxe2x80x9d defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host. The cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript. Thus, two sequences, such as a promoter and a xe2x80x9creporter sequencexe2x80x9d are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence. In order to be xe2x80x9coperably linkedxe2x80x9d it is not necessary that two sequences be immediately adjacent to one another.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest. This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (i.e. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography . . . ). In addition, the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies. The purified protein can be used for therapeutic applications.
The DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule. xe2x80x9cPromoterxe2x80x9d refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3xe2x80x2 direction) coding sequence. For purposes of the present invention, the promoter is bound at its 3xe2x80x2 terminus by the transcription initiation site and extends upstream (5xe2x80x2 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain xe2x80x9cTATAxe2x80x9d boses and xe2x80x9cCCATxe2x80x9d boxes. Prokaryotic promoters contain xe2x88x9210 and xe2x88x9235 consensus sequences which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences which serve as ribosome binding sequences during translation initiation.
As used herein, the designation xe2x80x9cfunctional derivativexe2x80x9d denotes, in the context of a functional derivative of a sequence whether an nucleic acid or amino acid sequence, a molecule that retains a biological activity (either functional or structural) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivatives or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid as chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term xe2x80x9cfunctional derivativesxe2x80x9d is intended to include xe2x80x9cfragmentsxe2x80x9d, xe2x80x9csegmentsxe2x80x9d, xe2x80x9cvariantsxe2x80x9d, xe2x80x9canalogsxe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of the subject matter of the present invention.
Thus, the term xe2x80x9cvariantxe2x80x9d refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, all these methods are well known in the art.
As used herein, xe2x80x9cchemical derivativesxe2x80x9d is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington""s Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
The term xe2x80x9callelexe2x80x9d defines an alternative form of a gene which occupies a given locus on a chromosome.
As commonly known, a xe2x80x9cmutationxe2x80x9d is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist. The result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d refers to a molecule having been separated from a cellular component. Thus, for example, a xe2x80x9cpurified proteinxe2x80x9d has been purified to a level not found in nature. A xe2x80x9csubstantially purexe2x80x9d molecule is a molecule that is lacking in all other cellular components.
In certain embodiments, it might be beneficial to use fusion proteins comprising the protein of the present invention, a part thereof or a derivative thereof. Non limiting examples of such fusion proteins include a hemaglutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions. In certain embodiments, it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused. Such protease cleavage sites between two heterologously fused polypeptides are well known in the art.
In certain embodiments, it might also be beneficial to fuse the protein of the present invention, a part thereof or a derivative thereof, to signal peptide sequences enabling a secretion of the fusion protein from the host cell. Signal peptides from diverse organisms are well known in the art. Bacterial OmpA and yeast Suc2 are two non-limiting examples of proteins containing signal sequences. In certain embodiments, it might also be beneficial to introduce a linker (commonly known) between the interaction domain and the heterologous polypeptide portion. Such fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like.
For certainty, the sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) interaction domain. It will be clear to the person of ordinary skill that whether an interaction domain of the present invention, variant, derivative, or fragment thereof retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
As exemplified herein below, the protein of the present invention, a part thereof or a derivative thereof, can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds. However, some derivative or analogs having lost their biological function may still find utility, for example for raising antibodies. These antibodies could be used for detection or purification purposes. In addition, these antibodies could also act as competitive or non-competitive inhibitors and be found to be modulators of protease activity.
The antibodies of the present invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(abxe2x80x2)2 fragment; the Fabxe2x80x2 fragments, Fab fragments, and Fv fragments.
Of special interest to the present invention are produced in humans, or are xe2x80x9chumanizedxe2x80x9d (i.e. non-immunogenic in a human) by recombinant or other technology. Humanized antibodies can be produced for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson et al., International Patent Publication PCT/US86/02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496. Reviews on humanized chimeric antibodies include Morrison, 1985, Science 229: 1202-1207 and Oi et al., 1986, BioTechniques 4:214.
In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, 1984, supra; Harlow et al., 1988, supra; and St. Groth et al., 1980, J. Immunol. Methods 35: 1-21. In general, techniques for purifying monoclonal antibodies are also well known in the art (Campbell, 1984, supra; Harlow et al., 1988, supra). Non-limiting examples of monoclonal antibody purification methods include ammonium sulfate precipitation, ion exchange chromatography and HPLC. Monoclonal antibodies can also be produced by bioreactor such as the hollow fiber cell culture system described in (the Unisyn instruction manuel). For example, using this hollow fiber membrane having a molecular weight cut off of 35.000, 1xc3x97108 cells of hybridoma are introduced into the bioreactor. The hybridoma can be grown in PFHM-11 media (GIBCO, BRL) with PEN/STREP (GIBCO/BRL). In certain embodiments of the present invention, it might be advantageous to provide the above-described antibodies as detectably labeled.
From the specification and appended claims, the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents. Further, the DNA segments or proteins according to the present invention can be introduced into individuals in a number of ways. For example, erythropoietic cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways, including intravenous injection. Alternatively, the DNA construct can be administered directly to the afflicted individual, for example, by injection in the bone marrow. The DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes.
For administration to humans, the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (i.e. DNA construct, protein, cells), the response and condition of the patient as well as the severity of the disease.
Composition within the scope of the present invention should contain the active agent (i.e. fusion protein, nucleic acid, and molecule) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Typically, the nucleic acids in accordance with the present invention can be administered to mammals (i.e. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington""s Pharmaceutical Science, 16th Ed., Mack Ed.). For the administration of polypeptides, antagonists, agonists and the like, the amount administered should be chosen so as to avoid adverse side effects. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
Other objects features and advantages of the present invention will become apparent upon reading of the following non-restrictive description of the preferred embodiments thereof given by way of example only with reference to the accompanying drawings and from the claims.